In hypokalemia or lowered extracellular K+ levels, human cardiac cells can become paradoxically depolarized inconsistent with the Nernst equation for K+. Such paradoxical depolarization has been observed for over thirty years. It is also crucial to the etiology of hypokalemia-induced cardiac arrhythmia. However, its molecular mechanism is not well understood. Ion selectivity of K+ channels is generally considered to be static and not changed in response to physiological stimuli. How K+ channels select K+ over other monovalent cations still remains an unsolved question. Particularly, ion selectivity and the selectivity filter of dimeric two-pore domain K+ channels (K2P) are less understood compared to those in tetrameric K+ channels. Our long-term goal is to understand physiological roles of K+ channels and molecular mechanisms of K+ channel function. The objective of this proposal is to explore functional roles of TWIK-1, the first cloned member of mammalian K2P channels in human cardiomyocytes in hypokalemia, and to characterize dynamic behaviors in ion selectivity and permeability of K2P channels. Based on studies in the past thirty years, the implications that are derived from these studies, and our preliminary studies on cloned TWIK-1 K+ channels, we hypothesize: 1) TWIK-1 K+ channels respond to challenges of lowered extracellular K+ levels and contribute to paradoxical depolarization in human cardiomyocytes in hypokalemia by changing ion selectivity and conducting inward leak Na+ currents; 2) K2P channels can adjust the conformations of the selectivity filter and exhibit dynamic behaviors in ion selectivity and permeability for monovalent cations. By employing standard methods and conventional approaches in the field of ion channels and electrophysiology, we will test these hypotheses in two specific aims: 1) Investigate how TWIK-1 K+ channels regulate the resting potential and action potential of human primary cardiomyocytes in both normal and hypokalemic conditions. 2) Study ion selectivity and permeability of K2P channels for small alkali metal ions and large organic monovalent cations in the absence of intracellular K+. The proposed research will demonstrate physiological roles of TWIK-1 K+ channels in human cardiomyocytes, describe a novel mechanism that regulates cardiac excitability, provide novel insights on the understanding of paradoxical depolarization in the heart in hypokalemia, and shed light on the etiology of hypokalemia-induced cardiac arrhythmias. It will also introduce the concept of dynamic ion selectivity of K+ channels under physiological conditions, provide evidence of a flexible K+ selectivity filter, which supports or supplements well-known hypotheses regarding ion selectivity of K+ channels, and improve the understanding of ion selectivity and the selectivity filter of K2P channels.